Abstract

Introduction: Systemic sepsis is the body's response to invading microorganisms is from bacteria and fungi. In the most severely ill patients are vitamin D deficient. Since vitamin D in innate and adaptive immune responses is essential for the proper functioning of antimicrobials. The aim of this clinical trial to evaluate the safety associated with vitamin D levels in patients with severe sepsis.

Methods: In this clinical trial, 60 patients with sepsis were divided into 3 groups of 20 and 20 cases of healthy people as a control group was considered. Serum levels of IL-10 and TNF-alpha, length of stay and mortality were recorded. The data was analysed.

Results: A statistically significant difference in mean age and sex distribution between the groups was observed. The patients showed a significant difference that indicates that vitamin D deficiency in the number of people admitted they were more than the other two groups were fatal. The mean levels of vitamin D, IL-10 and TNF levels in vitamin D deficiency showed significant differences between groups (P value=001/0).

Conclusion: The results and lack of complication in the use of vitamin D muscle, as well as ease of use and low cost of these medications can these drugs in improving patient safety sepsis and reduce hospital admissions and deaths and morbidity can be as adjunctive therapy in the treatment of patients with symptoms of sepsis can be used.

Keywords

Vitamin D levels, Immune status, Sepsis, Patient, Serum.

Introduction

It considers that sepsis is a response to an invasive
microorganism such as bacteria and fungi [1] and it could
frequently occur in patients with malignancy; in addition, this
condition is associated with highly significant mortality [2]. In
recent years, despite significant improvement in symptoms by
using appropriate treatments [3], successful treatment remains
a major challenge which is tightly associated with the rapid
removal of microorganisms from the body and supportive
measures [4,5].

Past investigations demonstrated that vitamin D deficiency
[6-10] commonly occurs in most severe disorders. Numerous
studies have shown that there is a correlation between lower
vitamin D levels and some conditions including sepsis,
increased the duration of hospitalization [11], high
inflammatory responses [12], and acute kidney damage
[13,14]. However, the exact mechanism of this condition is still
understood. It has suggested that due to the potential role of the active metabolite of vitamin D (1 and 25 dihydroxy-vitamin D)
on the host defense system [15].

Primarily vitamin D considers as a prohormone in calcium and
phosphorus homeostasis for proper neuromuscular function
and musculoskeletal health. This vitamin is available through
the diet or by exposing ultraviolet radiation to the sun. Then
the liver turns it into 25 hydroxyvitamin D and finally to an
active form of 1 and 25 dihydroxy vitamin D in the kidneys,
which increases the calcium intake in the intestines. Vitamin D
levels also fluctuate based on seasonal variations. Recent
studies have shown that vitamin D could perform actions
beyond its intrinsic form [16]. Most types of cells, including
immune cells, such as macrophages, B, and T lymphocytes,
have an intra-nuclear receptor of vitamin D that respond to
active metabolites 1 and 25 of dihydroxy vitamin D [17,18].
The active form of vitamin D inhibits the production of proinflammatory
cytokines including interleukin 2, 6, 8 and TNF-
α [19,20] and also increases the production of antiinflammatory
cytokines such as interleukin 10 [21,22], which can reduce the damage to the kidneys. Therefore, it could be
concluded that vitamin D is not only an alternative indicator
for determining the severity of the disease but also an
important factor in the incidence and severity of sepsis [23].
Since vitamin D is essential for proper antimicrobial function
in immune responses [24,25], this clinical trial aims to
investigate the effect of vitamin D on the immune response of
patients with severe sepsis.

Materials and Methods

Conditions and sampling

According to the incidence of 2% sepsis and 0.3% severe
sepsis in hospitalized patients, we used to survey all patients
with an early diagnosis of severe sepsis in Valiasr Arak
hospital. Also, we enrolled 20 healthy people of the
community, which were matched for age and sex, and other
criteria for inclusion with an average vitamin D level, and
considered them as a control group. Also, due to changes in the
seasonal pattern of vitamin D, which is the lowest during the
winter and spring, and in the summer and fall seasons, the
present study has been selected for the period of 6 months of
autumn and winter.

Study design

In this study, patients with sepsis (two criteria: temperature
between 36 and 38, heart rate more than 90, respiratory rate
more than 20 or arterial CO2 pressure more than 32, WBC
between 4000 and 12000, and the presence of a new source of
infection: (Blood pressure<60/90 or MAP<70 or BP>40 mmg,
mottled skin or capillary filling more than or equal to 3 s or
PaO2/FIO2<300, all of which are arterial hypoxia, DIC, acute
renal failure or urine flow<0.5 ml/kg/h or <45 mmol/L for at
least 2 h, heart failure, serum lactate>2, creatinine level
Serum>2 mg/dl or Cr>0.5 mg/dl, platelet counts below
100.000, liver failure based on bilirubin level>2 mg/dl or
INR>1.5 or PTT>60, acute pulmonary injury or ARDS, nonintestinal
sounds or ileus, which are not justified by other
causes of organ dysfunction) during the season fall and winter
visits to Vali-e-Asr Hospital in Arak were entered into the
study after receiving written consent and according to inclusion
and exclusion criteria. If necessary, after initial recovery and
simultaneously with the initiation of specific treatment for
these patients, Serum vitamin D levels were measured in their
kidneys (group A). According to the level of vitamin D,
patients were divided into 3 groups (groups A1: patients with
vitamin D deficiency (vitamin D levels<20 ng/dl or <50
nmol/L), A2: patients with vitamin D deficiency (level vitamin
D between 20 and 9/29 ng/dl or 50 and 72 nmol/L), A3:
patients with adequate levels of vitamin D (vitamin D>30 ng/dl
or >75 nmol/L). Also, 20 healthy people in the community,
which were matched for age and sex and other criteria for
inclusion and exclusion were considered as the control group
(group B). In this step, serum levels of interleukin 10 and TNF-
α were measured in both groups. Sampling was done through
the cubital vein. Then the samples were centrifuged at room temperature for 20 min and, according to the necessity of
measuring the level of interleukins during a session, samples
were stored in a refrigerator of 70°C in Immunology lab of
Arak University of Medical Sciences until complete collection
of samples. Then we measured the level of interleukin 10 and
TNF-alpha, and the results recorded. It should be noted that the
data recorded by an emergency specialist assistant.

Inclusion and exclusion criteria

Inclusion: 70>Age>18, and severe sepsis diagnostic criteria

Exclusion: Chronic kidney disease (Cr>2)

Chronic liver disease

Chronic heart failure

Previous gastrostomy or intestinal malabsorption

Calcium level ≥ 10

Receive vitamin D supplements in the last 7 days or antidepressant
drugs

History of parathyroid disease

Metabolic bone diseases, nephrolithiasis, sarcoidosis, ESRD

Imminent death

Pregnancy

Dissatisfaction with the company in the study

Statistical analysis

Data were analysed by SPSS22 software, Chi-square and t-test,
and ANOVA with repeated observations.

Results

This double-blind clinical trial was performed on patients with
sepsis. In this study, 60 patients with sepsis which referred to
Vali-Asr Hospital in Arak were divided into three groups:
vitamin D deficiency (below 20), vitamin D deficiency (20 to
30), and normal vitamin D levels (above 30) and 20 healthy
people were considered as control group. The mean age in
subjects with vitamin D deficiency was 62.75 ± 6.84, vitamin
D was 55.75 ± 10.8, and vitamin D was 54.7 ± 10.07,
respectively, and in The control group had a mean age of 42.4
± 8.35, which according to the P value, there was no significant
difference in mean age between the groups and were matched
in age (P value=0.6246, Table 1).

P value

Group

Age

SD

Mean

0/624

6/84

62/75

Vit D (less than 20)

10/80

55/75

Vit D (20-30)

10/07

54/70

Vit D (over 30)

8/35

42/40

Control

11/61

53/90

Total

Table 1: Relation between vitamin D and age.

The distribution of sex in the vitamin D deficiency group was 9
(45%) male, and 11 (55%) were female. In the sufficient
vitamin D group, ten were male (50%), and 10 were female
(50%). In the normal vitamin D group, 12 (60%) were male
and 8 were female. Also, in the control group, twelve (60%)
men and eight were women. According to the P value, there
was no significant difference in gender between the subjects
(Table 2).

P value/th>

Group/th>

Gender/th>

Percent/th>

Frequency/th>

0/715/td>

45%/td>

9/td>

Male/td>

Vit D (Less than 20)/td>

55%/td>

11/td>

Female/td>

50%/td>

10/td>

Male/td>

Vit D (20-30)/td>

50%/td>

10/td>

Female/td>

60%/td>

12/td>

Male/td>

Vit D (over 30)/td>

40%/td>

8/td>

Female/td>

60%/td>

12/td>

Male/td>

Control/td>

40%/td>

8/td>

Female/td>

53/75%/td>

43/td>

Male/td>

Total/td>

46/25%/td>

37/td>

Female/td>

Table 2: Distribution of gender including vitamin D levels.

The distribution of patients in the vitamin D deficiency group
was 12 cases discharged (60%) and 8 (40%) died, in the
vitamin D sufficient group, 18 (90%) were discharged and 2
(10%) died, and in the normal level of vitamin D, 17 (85%)
were discharged and 3 (15%) died. According to the P value,
there was a significant difference between the three groups of
participants in the design. This means that in the vitamin D
deficiency group, the number of hospitalized patients was more
than the others (Table 3).

P value

Group

Patients condition

Percent

Frequency

0/048

60 %

12

Discharged

Vit D (less than 20)

40 %

8

Died

90 %

18

Discharged

Vit D (20-30)

10 %

Died

85 %

17

Discharged

Vit D (over 30)

15 %

3

Died

78/33 %

47

Discharged

Control

21/66 %

13

Died

Table 3: Frequency distribution of patients’ condition.

The mean vitamin D level in the vitamin D deficiency group
was 11.28 ± 5.21 in the vitamin D deficiency group of 25.95 ±
2.74 and the normal vitamin D group was 37.75 ± 7.38 and in
the control group was 46.06 ± 10.87 There was a significant
difference between the groups in terms of P value (P
value=0.001, Table 4).

P value

Group

Vitamin D levels

Mean

SD

0/001

5/21

11/28

Vit D (Less than 20)

2/74

25/95

Vit D (20-30)

7/38

37/75

Vit D (over 30)

10/87

46/06

Control

14/94

30/26

Total

Table 4: Mean level of vitamin D.

The mean level of IL-10 in the vitamin D deficiency group was
5.03 ± 8.29 in the vitamin deficient group of 15.92 ± 7.70 and
the normal level of vitamin D was 21.78 ± 7.84 and in the
control group it was 53.23 ± 21.15 There was a statistically
significant difference between the groups (P value=0.000,
Table 5).

P value

Group

IL-10 level

Mean

SD

0/001

5/03

8/29

Vit D (Less than 20)

7/70

15/92

Vit D (20-30)

7/84

21/87

Vit D (over 30)

21/15

53/23

Control

20/94

24/83

Total

Table 5: Mean level of IL-10.

The mean TNF-a level in the vitamin D deficiency group was
19.10 ± 10.92 in the vitamin D deficiency group 11.67 ± 3.53
and the normal diet group 11.61 ± 3.60 and the control group
5.48 ± 2.7. According to P value, there was a significant
difference between the groups (P value=0.001, Table 6).

P value

Group

TNF-a level

Mean

SD

0/001

10/92

19/10

Vit D (Less than 20)

3/35

11/67

Vit D (20-30)

3/60

11/61

Vit D (over 30)

2/07

5/48

Control

7/69

11/96

Total

Table 6: Mean level of TNF-a.

The mean duration of hospitalization in patients with
hypothyroidism in the vitamin D deficiency group was 4.4 ±
2.47, in the vitamin D deficiency group was 2.31 ± 3.90 and in
the normal diet group was 3.65 ± 1.59, there was no
statistically significant difference between the studied groups
(p value=0.540, Table 7).

P value

Group

Hospitalization duration

Mean

SD

0/001

2/47

4/40

Vit D (Less than 20)

2/31

3/90

Vit D (20-30)

1/59

3/65

Vit D (over 30)

2/15

3/98

Control

Table 7: Mean level of hospitalization duration.

The average duration of hospitalization in patients without
vitamin D deficiency was 2.30 ± 5.33, 2.40 ± 4 in vitamin D
deficiency and 1.49 ± 3.88 in normal vitamin D group. There
was no significant difference between the groups (P=0.414,
Table 8).

P value

Group

Hospitalization duration

Mean

SD

0/001

2/30

5/33

Vit D (Less than 20)

2/40

4

Vit D (20-30)

1/49

3/88

Vit D (over 30)

2/12

4/29

Control

Table 8: Mean duration of hospitalization in patients without died one.

Discussion

This study aimed to evaluate the immune status of vitamin D in
patients with severe sepsis. Vitamin D could affect human
immunity, for example, by preventing excessive release of
cytokine and activating the integrated immune system. The
results of past studies revealed the role of vitamin D in
infectious diseases, but often limited to a small number of
patients.

To investigate the effect of vitamin D on the sepsis disease,
researchers reviewed the records of patients admitted to the
intensive care unit in Boston between March 1, 1998, and
January 2011. The 3386 adults were analysed by measuring the
amount of vitamin D from 1 year before admission. Women
accounted for 46% of the patients, 81% were white, and 67%
had a medical diagnosis. 69% of vitamin D measurements were
performed less than six months before admission to ICU [26].
According to the International Classification of Diseases, for
each five ng/ml increase in vitamin D levels before admission,
the ratio of sepsis decreased by 4%.

In sepsis, the cases with less than 30 ng/ml of vitamin D levels
before treatment, the rate of mortality was 1.6 times higher in
comparison to normal vitamin D level.

In this study, there was no significant difference in mean age
between the groups [9]. Also, the frequency distribution of
gender did not show a significant difference between the
groups.

Haan et al.’s meta-analysis study, concluded that vitamin D
levels below 50 were associated with an increase in risk of
infections, sepsis, death within 30 d, and death in the hospital
[27].

Leaf et al. conducted a study which shows that the primary
outcome of using cathelicidine protein in the first 24 h after
injection and the secondary outcome was the level of cytokines
and the marker of renal injury. In their study, they concluded
that administration of calcitriol did not increase the level of
cathelicidine in severely painful patients with sepsis, and also
had different effects on immune markers [14].

Amrein et al. indicated that most patients suffered from
vitamin D deficiency, and this was different in winter
compared with summer [28].

Jeng et al. concluded that there is a correlation between the
severity of the disease, vitamin D deficiency and vitamin Dbinding
protein levels in severely ill patients in comparison to
healthy. They also concluded that there is a positive
relationship between vitamin D level and cathelicidine LL-37
[9].

Frequency distribution of patients showed a significant
difference between the three groups in the study, which
indicates that the number of hospitalized patients who died in
vitamin D deficiency was more than the other two groups.
Mean vitamin D levels and interleukin 10 levels also showed a
significant difference between the studied groups. There was
no significant difference in the mean hospitalization time in
patients with dead patients, among the studied groups. Also,
there was no significant difference in the mean duration of
hospitalization in the patients without considering the deaths
among the patients.

Conclusion

According to the results of this study, use of vitamin D is an
easy and low cost; it can be used to improve the immune status
in sepsis patients and reduce hospitalization and mortality in
patients. It was used as an auxiliary therapy for treating
patients with sepsis symptoms. It should be noted that the
reduction in the hospitalization days, hospital infections are
reduced, treatment costs are reduced.